U.S. patent number 6,695,765 [Application Number 09/570,483] was granted by the patent office on 2004-02-24 for microfluidic channel embryo and/or oocyte handling, analysis and biological evaluation.
This patent grant is currently assigned to The Board of Trustees of the University of Illinois. Invention is credited to David J. Beebe, Ian K. Glasgow, Matthew B. Wheeler, Henry Zeringue.
United States Patent |
6,695,765 |
Beebe , et al. |
February 24, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
Microfluidic channel embryo and/or oocyte handling, analysis and
biological evaluation
Abstract
Microfluidic embryo scaled channels for handling and positioning
embryos provide the opportunity to evaluate and treat embryos in
improved manners Fluid flow is used to move and position embryos
within microfluidic channels and channel geometries may be used to
place embryos at specific locations. Surface properties and
compliance (deformation) properties of embryos are evaluated as a
predictor of viability. The microfluidic channels provide the
opportunity for fine controls of pressure to conduct various
evaluations at forces slightly below which damage to embryos is
known to occur.
Inventors: |
Beebe; David J. (Monona,
WI), Glasgow; Ian K. (Madison, WI), Wheeler; Matthew
B. (Tolono, IL), Zeringue; Henry (Madison, WI) |
Assignee: |
The Board of Trustees of the
University of Illinois (Urbana, IL)
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Family
ID: |
23110207 |
Appl.
No.: |
09/570,483 |
Filed: |
May 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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289137 |
Apr 8, 1999 |
6193647 |
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Current U.S.
Class: |
600/33 |
Current CPC
Class: |
A61B
17/435 (20130101); A61D 19/04 (20130101); B01L
3/502761 (20130101); C12M 21/06 (20130101); C12M
23/16 (20130101); C12M 29/10 (20130101); C12M
41/00 (20130101); B01L 3/502707 (20130101); B01L
2200/143 (20130101); B01L 2400/0457 (20130101); B01L
2400/0487 (20130101); B01L 2400/0688 (20130101); B01L
2400/086 (20130101) |
Current International
Class: |
A61D
19/00 (20060101); A61B 17/42 (20060101); A61B
17/435 (20060101); A61D 19/04 (20060101); A61B
017/43 (); A61D 007/00 () |
Field of
Search: |
;600/33,35 ;435/7.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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9115750 |
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Oct 1991 |
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WO |
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9322053 |
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Nov 1993 |
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WO |
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9322055 |
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Nov 1993 |
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WO |
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9747390 |
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Dec 1997 |
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WO |
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Other References
JM. Lim, B.C. Reggio, R.A. Godke, W. Hansel, "A Continuous Flow,
Perifusion Culture System for 8- to 16-Cell Bovine Embryos Derived
from In Vitro Culture", Theriogenology, vol. 46, pp. 1441-1450,
1996. .
J.A. Pruitt, D.W. Forrest, R.C. Burghardt, J.W. Evans, D.C.
Kraemer, "Viability and Ultrastructure of Equine Embryos Following
Culture in a Static or Dynamic System", Journal of Reproduction and
Fertility, vol. 44 (Supp.), pp. 405-410, 1991. .
C.L. Keefer, S.L. Stice, A.M. Paprocki, P. Golueke, "In vitro
Culture of Bovine IVM-IVF Embryos: Cooperative Interaction Among
Embryos and the Role of Growth Factors", Theriogenology, vol. 41,
pp. 1323-1331, 1994. .
P.C.H. Li and D.J. Harrison, "Transport, Manipulation, and Reaction
of Biological Cells On-Chip Using Electrokinetic Effects",
Analytical Chemistry, vol. 69, No. 8, pp. 1564-1568, 1997. .
S.J. Choi, I. Glasgow, H. Zeringue, D.J. Beebe, M.B. Wheeler,
"Development of Microelectromechanical Systems to Analyze
Individual Mammalian Embryos: Embryo Biocompatibility", Biol.
Reprod., vol. 58 (Suppl. 1), p. 96 (abstr.), 1998. .
K. Chun, G. Hashiguchi, H. Toshiyoshi, H. Fujita, "An Array of
Hollow Microcapillaries for the Controlled Injection of Genetic
Materials into Animal/Plant Cells", presented at Technical Digest
of Twelfth IEEE International Conference on Micro Electro
Mechanical Systems (MEMS '99), Orlando, FL, 1999, pp. 406-411.
.
I.K. Glasgow, H.C. Zeringue, D.J. Beebe, S.J. Choi, J.T. Lyman,
M.B. Wheeler, "Individual Embryo Transport and Retention on a Chip
for a Total Analysis System", presented at the Solid-State Sensor
and Actuator Workshop, Hilton Head Island, SC, 1998. .
I.K. Glasgow, H.C. Zeringue, D.J. Beebe, S.J. Choi, J.T. Lyman,
M.B. Wheeler, "Individual Embryo Transport and Retention on a
Chip", in Micro Total Analysis Systems '98; Proceedings of the TAS
'98 Workshop held in Banff, Canada, D.J. Harrison and A. van den
Berg, Eds. Boston: Kluwer Academic Publishers, pp. 199-202, 1998.
.
M.B. Wheeler, S.J. Choi, I.K. Glasgow, H.C. Zeringue, J.T. Lyman,
D.J. Beebe, "Development of Microelectromechanical Systems to
Analyze Individual Mammalian Embryos: Embryo Biocompatibility and
Individual Embryo Transport on Silicon A Chip", Arquivos da
Faculdade de Veterinaria UFRGS, Sociedade Brasileira de
Transferencia de Embraoes, vol. 26, No. 1, 1998 (Supl), p. 391.
.
K. Hosokawa, T. Fujii, I. Endo, "Hydrophobic Microcapillary Vent
for Pneumatic Manipulation of Liquid in .mu.TAS", in Micro Total
Analysis Systems '98; Proceedings of the TAS '98 Workshop held in
Banff, Canada, D.J. Harrison and A. van den Berg, Eds. Boston:
Kluwer Academic Publishers, pp. 307-310, 1998. .
"Microchip Arrays put DNA on the Spot", Science, vol. 282, Oct. 16,
1998, pp. 396-405. .
M. Lane and D.K. Gardner, "Selection of Viable Mouse Blastocysts
Prior to Transfer using a Metabolic Criterion", Human Reproduction,
vol. 21, No. 9, 1996, pp. 1975-1978..
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Primary Examiner: Shaver; Kevin
Assistant Examiner: Szmal; Brian
Attorney, Agent or Firm: Greer, Burns & Crain, Ltd.
Parent Case Text
REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of, and claims priority
under 35 USC .sctn.120 from U.S. application Ser. No. 09/289,137
filed Apr. 4-8, 1999, now U.S. Pat. No. 6,193,647.
Claims
What is claimed is:
1. A method for evaluating embryos, the method comprising steps of:
placing an embryo in an approximately embryo scaled fluid channel;
creating a fluid flow in the fluid channel; and evaluating an
embryo characteristic while an embryo is within the embryo scaled
fluid channel.
2. The method according to claim 1, wherein said step of creating a
fluid flow moves an embryo in the fluid channel, and said step of
evaluating an embryo characteristic comprises measuring the speed
at which an embryo moves in the fluid channel.
3. The method according to claim 1, wherein said step of creating a
fluid flow moves an embryo in the fluid channel, and said step of
evaluating an embryo characteristic comprises measuring the
distance an embryo moves in the fluid channel.
4. The method according to claim 1, wherein said step of evaluating
comprises obtaining a fluid sample from the fluid channel
downstream of an embryo in the fluid channel and conducting a
chemical analysis of the fluid sample.
5. The method according to claim 1, wherein the fluid channel
includes a constriction sized to deform an embryo as it passes
through and said step of evaluating evaluates a tendency of an
embryo to return its shape after passing through the
constriction.
6. The method according to claim 1, wherein the fluid channel
includes a constriction sized to prevent passage of an embryo, said
step of creating creates a fluid flow to move an embryo to the
constriction and then deform it slightly by fluid pressure for a
short period of time, and said step of evaluating evaluates a
tendency of an embryo to return its shape after being deformed for
the short period of time.
7. A method for treating embryos, the method comprising steps of:
placing an embryo in an approximately embryo scaled fluid channel;
creating a fluid flow in the fluid channel; and treating an embryo
to alter a characteristic of the embryo while it is positioned
within the fluid channel.
8. The method according to claim 7, wherein said fluid channel
includes a series of constrictions including protrusions having
gradually less space therebetween, and said step of treating
comprises manipulating the fluid flow in the fluid channel to pass
an embryo through one or more of the constrictions and to remove
cumulus.
9. The method according to claim 8, wherein a last one of the
series of constrictions is sized to block passage of the embryo,
and said step of treating comprises manipulating the fluid flow in
the fluid channel to pass an embryo through other ones of the
series of constrictions and then manipulating the fluid flow to
suck off cumulus when an embryo is positioned at the last one of
the series of constrictions.
10. The method according to claim 7, wherein the fluid channel
includes a constriction sized to prevent passage of an embryo, said
step of treating comprises altering the fluid in the fluid channel
to pass an acidic solution over an embryo positioned at the
constriction to remove zona pellucida.
11. A microfluidic embryo handling device comprising: an embryo
transport network having a biological medium for movement of
embryos inserted therein, said transport network including an
approximate embryo scaled embryo fluid channel and an opening for
delivering fluid into the transport network; and a t-junction in
the transport formed at the intersection of two fluid channels, and
a second opening for delivering fluid into the transport network at
a separate fluid channel location.
12. The device according to claim 11, further comprising a well in
said fluid channel formed to hold an embryo during periods
predetermined flow rate within said fluid channel and allow escape
of an embryo during periods of lower than said predetermined flow a
rate within said fluid channel.
13. A microfluidic embryo fertilization device comprising: an
embryo transport network having a biological medium for movement of
embryos inserted therein, said transport network including an
approximate embryo scaled embryo fluid channel and an opening for
delivering fluid into the transport network; and a T-junction in
the transport network formed at the intersection of two fluid
channels, and a second opening for delivering sperm into the
transport network at a separate fluid channel location.
Description
FIELD OF THE INVENTION
The present invention generally concerns handling of embryos. The
invention also concerns handling of oocytes (prefertilized
embryos), and eggs. Embryo, as used herein, therefore encompasses
oocytes, and eggs as well as fertilized embryos. The invention more
specifically concerns microfluidic handling of embryos for
culturing, manipulation, and analysis.
BACKGROUND OF THE INVENTION
Technology assisted reproduction techniques in which embryos are
handled independently from their mammalian biological source are
growing in importance and frequency of use. Such techniques have
great direct benefit to persons unable to have babies through
unassisted sexual reproduction. The agricultural industries also
increasingly rely upon such assisted reproduction techniques.
Embryo manipulation is used in livestock reproduction to control
such things as the faster genetic evolution of cattle and
permitting the genetic characteristics of a single exceptional cow
or bull to be passed on to far greater numbers of offspring than
would be possible through unassisted sexual reproduction.
Livestock embryo manipulation is becoming more routine due to the
development of gene manipulation, cloning, and in vitro
fertilization (IVF) techniques. The overall goal of embryo
manipulation in livestock is to increase production efficiency,
especially with regard to reproduction, milk production or
production of specific milk components, lean tissue growth with
reduced fat content and decreased susceptibility to specific
diseases. Embryo transfer is also used to introduce or rescue
valuable germplasm and propagate rare breeding animals such as
endangered exotic species.
Expense and relatively low success rates place significant burdens
on the use of these assisted reproduction techniques for humans as
well as livestock. In human reproduction such expense and failure
adds emotional as well as economic burdens. In addition, safeguards
against failures often result in unwanted or unmanageable multiple
births, as well as additional stored embryos which require
maintenance and additional difficult decision making at some later
point in time. Expense is the primary concern in livestock
reproduction.
Failure rates in reproduction techniques as well as testing and
other embryo handling techniques are attributable primarily to the
significant handling and manipulation of embryos in executing these
techniques. Animal reproductive technologies have advanced in
recent years, but the physical tools used in animal reproduction
have not changed significantly. Fine-bore glass pipets are still
one of the basic tools of the embryologist. Using standard petri
dishes, procedures such as in vitro maturation of eggs (IVM), in
vitro fertilization, and embryo culture (EC) require picking up and
placing individual eggs and embryos several times for each
procedure.
Such handling and movement from one petri dish to another provides
significant potential for damage or contamination. Perhaps more
important, though, is the failure of a stationary embryo in a petri
dish to simulate the corresponding natural biological reproduction
condition. Some efforts have been made to move embryos in petri
dishes via agitation of the dish, but this is a haphazard approach.
Expense is also created here due to the relatively large amount of
biological medium required for the manual petri dish conventional
embryo handling methods. Bovine embryos are individually handled
with pipets and large, expensive manipulators. Large quantities of
biological medium including growth agents for human embryo
culturing renders the corresponding in vitro procedure even more
expensive. Livestock growth factors, for example, have costs
exceeding $200 per 50 .mu.g.
Such static culture systems also fail to allow for changing the
milieu in the culture medium as the embryo develops. Current
culture systems with flowing medium have culture chambers as small
as 0.2 to 0.5 ml. However, the culture volumes are greater than
needed and medium is replenished too quickly. The endogenous growth
factors that enhance development are diluted out and washed away.
The large volumes of medium required substantially increase costs
when expensive growth factors, such as IGF-II ($200 per 50 .mu.g)
are used. In addition, known systems cannot track individual
embryos.
Conventional handling techniques also provide limited ability to
evaluate embryos. An ability to evaluate pre-implantation embryos,
including embryos, pronuclear zygotes, and oocytes, would provide a
better success rate for implantations. Currently, the morphology of
most embryos is evaluated prior to their transfer into a recipient.
Morphology examination will sort out embryos with gross defects but
is not a highly reliable indicator of viability. Chemical
monitoring over a period of time has been used, but requires
numerous measurements over a period of time. The result is a much
better predictor of viability, on the order of 80%, but many
embryos fail to survive the monitoring process. This requires use
of additional embryos. This results in multiple births, other
complications, and entails additional labor costs.
Conventional techniques also provide harsh methods for removal of
the zona pellucida, which is a critical step in the making of
chimeric embryos. Conventionally, an embryo is mouth pipetted from
one tissue culture dish containing the culture media, into a
culture dish containing an acidic media. The embryo is left in the
media for a period of time (tens of seconds) then mouth pipetted
into a dish containing fresh culture media. The embryo is then
flushed in and out of the pipette a few times to quickly disperse
all of the acidic media and minimize damage to the cell membranes.
The opening of the mouth pipette is about the same size as the
embryo, and the flushing therefore causes sheer stresses on the
embryos. The imprecision of mouth pipetting therefore provides
ample opportunity for damage.
Thus, there is a need for an improved embryo handling device and
method which addresses problems in known embryo handling
techniques. An improved embryo handling device and method should
provide for an improved simulation of natural conditions. It should
also provide a building block upon which larger and/or more
powerful and accurate instruments may be based, such as embryo
culturing systems, embryo analysis systems, embryo storage systems,
and similar systems. There is a further need for improved
evaluation of embryo viability.
SUMMARY OF THE INVENTION
These needs are met or exceeded by the present microfluidic embryo
handling device and method. The invention simulates biological
rotating of embryos. An embryo fluidic channel moves an embryo
inserted therein with fluid, and is sized on the same scale as the
particular type of embryo or embryos to be handled. The sizing and
fluid communication produces a simulated biological rotating of
embryos. In addition, the fluid flow with and around the embryo or
embryos prevents stagnation, reducing the likelihood of the embryo
or embryos developing injuries that may be analogous to "bed
sores".
The invention also permits the biological medium fluid to be
altered gradually, having significant advantages compared to
repeatedly manually transferring an embryo from one medium to
another medium in a pipet or petri dish. Gradual changes avoid the
shock from sudden changes in local environment. The microfluidic
system of the invention further permits the co-culturing of an
embryo with other embryos, co-culturing of an embryo or embryos
with cells upstream of the embryo(s), and maintenance of a separate
control culture that shares a common biological medium with a
subject embryo(s) thereby ensuring that test embryos see the same
environmental conditions as the subject embryo(s).
Other aspects of the invention concern specific uses of the broader
principles of microfluidic embryo handling to manipulate, evaluate
and position embryos. One aspect concerns the use of a gradual
series of constrictions to remove surrounding material from an
embryo. This has been demonstrated to remove surrounding cumulus
from oocyte. A first few constrictions cut the cumulus, which can
then be sucked off of the oocyte in a final small constriction
which is sized to prevent passage of the oocyte.
Embryo evaluation is also realized in accordance with the basic
invention principles. In a preferred evaluation, surface properties
and compliance (deformation) properties of embryos are evaluated.
The microfluidic channels provide the opportunity for fine controls
of pressure to conduct various evaluations at forces slightly below
which damage to embryos is known to occur. Measurement of the
distance and/or speed which embryos roll in a same pressure
gradient microfluidic channel provides information, with healthy
embryos traveling slower or a shorter distance as they demonstrate
more stiction to channel walls. Positioned at a constriction,
healthy embryos also appear to deform less than unhealthy embryos
that are more readily pulled into a constriction. In addition,
healthy embryos appear to resume their shape better.
Fluid from microfluidic channels is easily collected downstream
without altering the embryo environment, providing a better
opportunity for chemical analysis of fluid chemical analysis than
convention manual handling and sampling techniques. In addition,
all of the fluid collected from a microfluidic channel has passed
over the embryo. This provides better evaluation information than
fluids stagnant around an embryo in a Petri dish. Through the
invention, medium can continuously or periodically pass over
embryos and be collected downstream, eliminating additional
handling required in a petri dish technique. The invention provides
more consistent fluid samples since fluid can be repeatedly
collected in the same manner, whereas samples taken from petri
dishes may vary based upon placement of the pipette that suctions
medium, i.e., how far or how close to an embryo.
Use of clear channel sections allows for many types of optical
analysis. Stains or dyes may be added for visual inspection at
clear sections. Clear sections also provide the opportunity to use
image analysis devices, since the microfluidic channels may be
configured to precisely position an embryo at a location for
analysis by imaging equipment.
Precise position of embryos using channels and constrictions of the
invention, and/or flow manipulation, further enables an improved
method for zona pellucida removal of mammalian embryos. Embryos are
moved through flow to a precise location where lysing agent can be
washed over the embryo to achieve zona removal.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects, features and advantages of the invention will be
apparent to artisans who read the detailed description and
reference the accompanying drawings, of which:
FIG. 1 shows a cross section of a preferred microfluidic embryo
handling device constructed in accordance with the present
invention;
FIG. 2(a) is a top view showing a preferred narrow microfluidic
channel constriction for embryo positioning;
FIG. 2(b) is a cross-sectional view of an alternate preferred
shallow microfluidic channel constriction for embryo
positioning;
FIGS. 2(c) and 2(d) are schematic views of an alternate preferred
fluid dynamic constriction;
FIG. 2(e) is a schematic view of an alternate preferred fluid
dynamic constriction;
FIG. 2(f) illustrates an alternate preferred mechanical
constriction geometry;
FIG. 2(g) shows a particular flow pattern in the FIG. 2(e) fluid
dynamic constriction useful, for example, for zona treatment;
FIG. 2(h) shows another flow pattern useful for delivering a
discrete fluid to an embryo;
FIG. 2(I) shows another flow pattern useful for delivering a
discrete fluid to an embryo;
FIG. 3(a) is a perspective view of a preferred gravity flow driven
microfluidic culturing and testing device constructed in accordance
with the present invention;
FIG. 3(b) is a schematic cross-section of microfluidic channel for
a zona pellucida removal procedure of the invention;
FIG. 4 is a block diagram of an embryo analysis device constructed
in accordance with the present invention;
FIGS. 5(a)-5(c) illustrate preferred embryo microfluidic channel
insertion and removal structures in accordance with the present
invention;
FIGS. 6(a)-6(b) illustrate a preferred culturing device constructed
in accordance with the present invention;
FIGS. 7(a) and 7(b) illustrate portions of constricted channel
useful for cumulus removal from oocytes;
FIGS. 8(a) and 8(b) illustrate a complete prototype device for
cumulus removal;
FIGS. 8(c)-(f) illustrate steps used in experiments with the FIGS.
8(a) and 8(b) prototype to remove cumulus from an oocyte;
FIGS. 9(a)-9(d) illustrate a deformation evaluation of an embryo
used as an indicator of embryo viability; and
FIG. 10 illustrates a preferred microfluidic channel configuration
for fluid analysis or sampling in accordance with the
invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a microfluidic embryo handling
device which reduces stress to embryos handled outside their
natural biological host. The device and method reproduce simulated
biological rotating of an embryo through fluid assisted movement in
a channel that encourages embryo slipping and rotating. Rotating,
as used herein, may include complete rotation or partial rotation.
Partial rotation might also be referred to as a rocking motion.
Referring now to FIG. 1 shown is a cross-section of a microfluidic
embryo handling device 10 including a embryo transport network 12
formed at least in part by a generally embryo scale channel 14. An
embryo 16 in the channel 14 will move with fluid flow in the
channel 14, while the close dimensions of the channel cause the
embryo 14 to move with a simulated biological rotating motion.
Channels up to ten times the embryo size have been used to create
rolling and slipping. In biological hosts, developing embryos in
their initial stages of development move toward the uterus to which
they will attach with a rotating and slipping motion. The
microfluidic channel 14 produces a simulation of such motion.
Sizing of a channel is important to establish the biological
rotating. Height is the critical dimension, and it has been found
that heights up to about three times the diameter of an embryo
induce the rotating. This ratio may be determined to vary somewhat
because fluid flow also plays a role, but the three to one maximum
ratio has been found to produce the rotating. It will be
appreciated that the channel width is less important. The width may
be selected arbitrarily. Thus, if embryos are to be kept in single
order, then the width would be less than twice the embryo diameter.
If more embryos are desired, larger width channels may be used.
Networks of the channels 14 provide a means to culture embryos, as
well as to move and place embryos to desired locations. During its
initial stages of development, the size of most mammalian embryos
remain generally constant during the first few days after
fertilization. Thus, the size of the channels 14 provide no
impediment to culturing an embryo therein. Advantageously, the
embryo 16 may be kept moving and/or may have a continuous or pulsed
fluid flow passed around it to avoid potential detrimental
biological effects on the embryo 16.
A preferred exemplary construction of a device 10 including a
channel is also illustrated in FIG. 1. The microfluidic channel 14
may be formed by any suitable micromachining technique into a
suitable material, such as a silicon wafer 18. The material chosen
must be capable of being sterilized and should not pose a
biological threat to embryos. The channel(s) 14 of the device are
sealed through a cover 20. Forming the cover of glass or other
transparent material allows convenient visual monitoring of embryos
in channel(s) 14. A bonding agent 22 bonds the cover 20 to the
wafer 18. Additionally, the material of the cover could be
formulated to shield harmful radiation from the embryo(s) in
channel(s) 14.
Unlike other cells that tend to float in a fluid medium, the
relatively large and heavy embryos sink to the bottom of the
microfluidic channels 14. Typical mammalian preimplantation embryos
of interest are 90 to 180 .mu.m diameter spheres. In each embryo, a
membrane surrounds each cell (blastomere) and the zona pellucida, a
glycoprotein membrane or shell, surrounds the entire cell mass. The
cells divide several times during the first few days after
fertilization, the volume of the embryo remains constant and an egg
may be fertilized and cultured to a blastocyst in the same device
constructed based upon the principles of the invention. The
blastocyst is the final stage before an embryo implants in the
uterus.
Also important to production of such a device and similar devices
is the ability to handle individual embryos, or small numbers of
embryos. Positioning embryos to given locations, moving to
alternate locations, and maintaining constant or changing
biological conditions around the embryo(s) are abilities provided
by basic principles of the present invention, and permit the
construction of fertilization, culturing, testing, and other
devices which rely on some or all of those abilities. For
continuous movement of an embryo through a culture period of time,
long channels may be created, or a loop may be formed. Alternately,
a parking of an embryo may occur at a culturing station like those
shown in FIGS. 6(a) and 6(b). A compartment or channel of limited
size may also be used to roll an embryo back and forth therein by
changing fluid flow, as will be further discussed with respect to
FIGS. 6(a) and 6(b).
Accurate positioning of individual embryos is provided by the
invention through the use of constrictions, preferred examples of
which are shown in FIGS. 2(a) and 2(b). FIG. 2(a) is a top view of
a cross section of a narrow constriction 24 formed in a
microfluidic channel 14. There are many reasons such an accurate
positioning may be desirable in an embryo handling device 10.
Analysis instruments built into the device may require an embryo to
be precisely positioned at electrodes, a photodetector, the focal
point of a microscope, or other similar sensing device.
Transporting an embryo to the constriction 24 permits such required
positioning without resort to feedback systems. An embryo 16 is
freed from the constriction 24 simply by reversing the flow of
biological fluid medium 30. Even when held at the constriction, an
embryo 16 experiences a flow of biological fluid medium around it
since fluid 30 will flow past it and through the constriction 24.
This is advantageous since an embryo in stagnant fluid has an
increased potential to develop "bed sores", a suspected but yet
unproven explanation for low success rates in embryo handling
technology.
Sidewall portions 28a, 28b of the microfluidic channel 14 constrict
it at a desired location to prevent passage of an embryo 16
therethrough. The constriction 24 does not completely close the
microfluidic channel 14 so that fluid biological medium 30 may pass
an embryo 16 positioned at the constriction 24. FIG. 2(b) shows a
side cross-section of an alternate shallow constriction 26 where
the fluid biological medium 30 is similarly able to pass when an
embryo 16 is positioned at the constriction. Other shapes of
constriction are also possible. Generally, any shape which prevents
passage of an embryo 16 while simultaneously allowing fluid flow
through the constriction, e.g., asymmetric shapes and comb-like
fibers, is acceptable to position embryos in a device 10 according
to the invention. It is preferred that the constriction be sized
such that positioning of an embryo prevents the embryo from passing
without an increased pressure from the fluid pressure used in a
device 10 to move embryos. Constriction length should also be kept
small enough to avoid fluid control problems since the constriction
portion of a microfluidic channel will have much higher fluidic
resistance per unit length than unrestricted portions of the
microfluidic channels 14.
Fluid dynamics within microfluidic channels 14 may also be used to
position embryos. Whereas the constrictions of FIGS. 2(a) and 2(b)
are physical constrictions, an effective fluid dynamic constriction
is realized in FIGS. 2(c)and 2(d) without a physical barrier to
passage of an embryo. In FIGS. 2(c) and 2(d), an increased depth
microfluidic channel well portion 14a defines a position where an
embryo 16 may be intentionally held by control of fluid dynamics,
i.e., the flow over and through the well portion 14a. In FIG. 2(c)
high laminar or nonlaminar flows will cause an embryo 16 to stay in
the well portion because flow separation occurs at the leading edge
of the well portion 14a. During (lower) flow rates, the fluid
stream lines follow the contour of the microfluidic channel 14,
including the well portion 14a, to sweep an embryo out of the well
portion. Predetermined flow rates to respectively hold and sweep
out an embryo can be calculated for particular well geometries and
sizings, or may be determined experimentally. FIG. 2(e) shows an
alternate strategy for positioning an embryo 16. In FIG. 2(e), a
T-junction 14b is formed at the intersection of microfluidic
channels 14. In a balanced fluid flow condition, as illustrated in
FIG. 2(e), there will be no flow at the embryo's position,
permitting it to remain in position. Though none is illustrated, an
indent or other small physical shape might enhance stability of the
embryo 16 in FIG. 2(e). Another constriction geometry is shown in
FIG. 2(f).
With the geometries and flows of FIGS. 2(a)-2(f) it is possible to
position an embryo at a precise location within a network of
microfluidic channels. This affords an opportunity for visual
inspection, embryo removal, embryo testing, analysis of the embryo
by imaging or other devices and treatment of an embryo. Many
mechanical manipulations of the embryo can accordingly be effected,
and any treatment, analysis, or manipulation that would benefit
from such accurate positioning an moving of the embryos, and the
ability to manipulate and control the flow environment therefore
benefits from application of the invention.
A specific exemplary treatment technique using constrictions and
microfluidic channel flows to position an embryo in a device like
the FIGS. 2(a)-2(e) devices is zona pellucida removal from an
embryo. The zona pellucida is a glycoprotein matrix surrounding
embryonic cells. A chimera, a single animal with two DNA sets, is
made by removing the zona pellucida and bringing the separate
embryonic cells together. Zona thinning or removal is also
important to transgenic procedures, IVF, and cell biopsy. A parking
location is defined for zona removal, see, e.g., FIG. 2(f). The low
aspect ratio, on the order of 0.01, for the top section of
constriction in FIG. 2(f) may require support to avoid collapse. In
a prototype device, this was achieved with small PDMS
(polydimethylsiloxane) posts surrounding the constriction region.
The constriction region against which an embryo, or multiple
embryos, will rest may be shaped to bring embryos together as will
be necessary for chimera formation. A v-shaped resting surface will
bring embryos together in such a fashion. Upstream of a
constriction like that shown in FIG. 2(f), microfluidic channels
are configured such that a controlled wash of acidic solution can
be caused to flow a parked embryo or embryos. For example, an
acidic inlet can t-junction into a main flow leading to the parking
area of FIG. 2(f). A lysing plug, for example, provides acidic
solution into the main channel near and upstream of the
constriction for a short period to achieve zona removal. With the
microfluidic channels, precise control of the flow is achieved for
a required period of time. In a prototype device, syringe
connections to a main microfluidic channel including the
constriction and a "T" intersection channel were used to control
embryo positioning and flows over positioned embryos. Syringes
offer precise control of fluid, and computer controlled
microsyringes will add precision to flow control and timing.
An alternate mechanical method of removing a zona involves
mechanical damage to the zona. This is achieved, for example, by
passing the zona through a microfluidic channel of the invention
including a mechanical structure to nick or cut the zona. The
precise fluid control of the invention also creates the possibility
of a laminar flow method to "nick" a zona. In the "T" junction of
FIG. 2(e) two separate flows of two different solutions meet at the
"T" and flow into a single channel, as shown in FIG. 2(g), where a
dotted line indicates separation of flow. These flows may be kept
separate in the single channel by laminar flow. One flow (on the
left) contains, for example, acid for zona thinning. Driving
pressures control the lateral position of the interface between the
two solutions such that the zona of an embryo 16 is "nicked" by the
acid.
The method for delivering discrete fluid to an embryo for zona
removal is also applicable to discrete delivery of other fluids,
for example, spermatozoa. Alternate multiple flow arrangements are
shown in FIGS. 2(h) and 2(I) where a cross junction of microfluidic
channels is shown. In FIG. 2(h), a main fluid flow A is controlled
to prevent entry of fluid B except for a limited time to form a
plug of the B fluid. A plug of fluid B is formed through fluid
control, i.e., a burst of increased pressure in the B flow, or a
temporary reduction or stop in the A flow. A created plug of fluid
B then flows with flow A over the embryo 16. Sizing of the channels
used to deliver flow B determines sizing of a plug of fluid B that
can be formed. This configuration has been used to deliver an acid
for zona removal, but could deliver other fluids in similar
fashion. FIG. 2(I) has the same geometric configuration, but uses
fluid flow to continuously deliver flow B in a portion of a
channel. This is similar to the control in FIG. 2(g).
A culture and test device 31 including a constriction like that
shown in FIG. 2(a) for positioning an embryo is illustrated in FIG.
3(a). The device 31 has fluid flow in a network 32 of microfluidic
channels 14 driven by gravity based upon levels of fluid 30 in a
plurality of fluid reservoirs 34. Any suitable means for driving
fluid 30 is contemplated as being compatible with the general
principles of the invention, e.g. pumping, but the gravity method
illustrated in FIG. 3(a) is preferred for its simplicity and
efficiency. Directions of flows are controlled simply by levels of
fluid in reservoirs 34. Thus, for example, an embryo 16 held at a
constriction 24 for culturing or examination by a suitable
instrument is positioned by first setting fluid levels to cause its
travel from inlet port 36 to constriction 24, and is released when
fluid flow is reversed through the constriction 24. Removal of the
embryo 16 is accomplished by causing fluid flow to move it to exit
port 38.
During movement through the microfluidic channels 14 of the network
32, the embryo(s) roll and slip to simulate natural movement of
embryos toward a uterus in a mammalian host, as discussed above.
This desirable manner of moving may be aided by a suitable
surfactant such as BSA (bovine serum albumin). The surfactant will
help to promote some slippage of the embryo as it rolls.
FIG. 3(a) also illustrates an additional advantage of the
invention, in the provision of a parallel additional microfluidic
handling and culturing device 31a. The additional device 31a has a
structure similar to that of device 31, but may have fewer or even
a single microfluidic channel. Ideally, the structure is the same.
The important feature of the device 31a is that it shares a common
fluid source with inlet port 36 and outlet port 38 of primary
device. Embryo(s) handled in the device 31a are isolated
biologically from embryo(s) in primary device 31, but experience
the same biological conditions through sharing the same fluid
source, pressure and/or the same biological medium condition. In an
exemplary use, the additional device 31a therefore might form an
important control culture in which development or lack of
development of test embryo(s) could confirm suitability or
unsuitability of conditions created in the primary device 31.
FIG. 4 is a block diagram of an embryo analysis device. In the FIG.
4 device a network 32 of microfluidic channels 14 moves embryos to
one or more analysis stations 40a, 40b or 40c. Embryos are
positioned at a given analysis station through constrictions like
those described above. The analysis stations may include any
instrument capable of obtaining information concerning an embryo,
with the constriction being formed to position embryos at the
proper sensing point for the particular instrument used in an
analysis station. Embryos are moved out of the device through one
or more exit ports 38a, 38b, which might alternately lead to a
culturing station in the form of a parking area for an embryo, an
additional length of microfluidic channel 14, or a microfluidic
channel loop for continuous movement of an embryo during
culturing.
Inlet and outlet ports used in devices of the invention may
comprise any conventional manner or structure for embryo insertion
or removal. However, additional preferred structures for insertion
and removal are shown in FIGS. 5(a)-5(c). In FIG. 5(a), a well 42
which is in fluid communication with a microfluidic channel 14 is
used. Fluid in the well 42 preferably also comprises a gravity feed
which helps drive microfluidic flow in the channel 14. An embryo 16
is placed in the well 42 and moves into the channel 14 with
biological medium, or simply sinks unaided into the channel 14 if
no flow condition is created. A second similar well 42 may be used
to remove an embryo using a pipet 44 or similar device, which might
also be used for insertion. In FIG. 5(b), a hanging drop 46 at the
end of a channel 14 is used for insertion and removal. The hanging
drop 46 is held by surface tension. After embryo insertion, fluid
may be added at that point, or the embryo may be sucked in by fluid
flow in the device. Alternately, the device may be inclined to
promote embryo movement away from the hanging drop 46. In FIG.
5(c), a funnel shaped hole 48 in direct communication with channel
14 is used for insertion and removal. The funnel shape aids
positioning of a pipet 44 or similar device. Surface tension at a
small diameter hole 48 will prevent fluid from leaking out, but the
pressure in channel 14 must not exceed the point that would defeat
surface tension and cause fluid to leak out. Inserted embryos will
sink into the channel 14, while removal may be accomplished by
drawing fluid from hole 48 when an embryo approaches. Of course,
any of the FIG. 5 techniques may be combined with each other or
conventional techniques for insertion and removal in a given
handling device. In addition, the wells 42 or holes 48 may be
covered by a removable cover or flap as protection against
contamination and/or evaporation.
Referring now to FIGS. 6(a) and 6(b), an embryo culturing device 50
according to the present invention is shown. Fluid medium flow in
the culture device 50 is in either direction between a medium inlet
52 and a medium outlet 54. The device includes a number of traps or
compartments 56. As best seen in FIG. 6(b), the traps 56 comprise
deep regions separated by shallow regions 58. Fluid flow between
inlet 52 and outlet 54 is over shallow regions and through deep
regions to move embryos back and forth within the deep region
compartments 56. Embryos are inserted and removed through access
holes 60, which may be formed by any of the preferred methods in
FIGS. 5(a) through 5(c). In the device 50, artisans will thus
appreciate that embryos may be moved back and forth within
compartments 56 to simulate biological rotating, may experience the
same medium conditions as other embryos within the culture, and may
be easily removed and inserted. Though FIGS. 6(a) and 6(b) show a
top loading embodiment for placing embryos within the compartment,
the device will also work in a bottom loading arrangement,
essentially inverted from that shown in FIGS. 6(a) and 6(b). In
such a bottom loading arrangement, the embryos will still be held
in the deep portions but cannot pass the shallow portions. An
alternate embodiment might comprise a gap in place of shallow
constrictions where embryos cannot pass through the gaps but fluid
flow may occur therebetween and the depth of the gaps may be the
same as those of the embryo holding compartments.
Prototype devices like that shown in FIG. 3(a) have been produced
and tested. Typical prototypes are described here for the sake of
completeness. Artisans will appreciate that the manner of
fabricating the prototypes may be accomplished by any other
convention micro fabrication techniques. Artisans will also
appreciate that production device manufacturing may differ
significantly, and that specific numerical dimensions and
conditions of the prototype devices do not limit the invention in
the breadth described above.
In typical prototype channels, a pressure gradient of 1 Pa/mm
causes the medium to flow on the order of 10.sup.-10 m.sup.3 /s
(100 nl/s), with an average speed of 1 to 2 mm/s. Under these flow
conditions the embryos roll along the bottoms of the channels;
traveling at speeds ranging from 1/3 to 1/2 that of the fluid that
would otherwise be in the same region of the channel. By
manipulating the pressure at the wells connected to the ends of the
channels, the embryos can be transported to (and retained at)
specific locations including culture compartments and retrieval
wells. Embryos fill a considerable portion of the channel, thereby
greatly altering the flow of medium. The flow of medium through the
channels is laminar.
Networks of prototype microfluidic channels have been fabricated in
a device like that shown in FIG. 3(a) by etching trenches in 3-inch
<100> silicon wafers, and then bonding glass covers to form
channels. Devices including microfluidic channels have also been
made by micromolding techniques in elastomers. Other plastics and
techniques are also likely to be suitable, including, for example,
injection molding of thermoplastic materials. Typical channel
networks contain several branch microfluidic channels that
intersect near the center of the device. The branches, which range
from 1.5 to 2.5 cm in length, are 160 to 200 .mu.m deep and 250 to
350 .mu.m wide at the top. A first step in producing prototype
devices involves patterning silicon nitride (SiN) coatings on using
conventional photolithography techniques. The microfluidic channels
are anisotropically etched with a potassium hydroxide (KOH)
solution. Access holes in the glass covers are drilled, either
conventionally using carbide tipped bits or ultrasonically. Glass
covers are bonded to the wafers using UV curable epoxy (NOA 61,
Norland Products, Inc, New Brunswick, N.J.) or Pyrex 7740 covers
are anodically bonded to the wafers using 500V in a 450.degree. C.
environment. The nitride coatings are removed using buffered oxide
etchant (BOE) before anodic bonding. Glass wells are bonded to the
glass cover at the end of each branch of the channel network with
either an epoxy (Quick Stick 5 Minute Epoxy or 5 Hour Set Epoxy
Glue; both from GC Electronics, Rockford, Ill.) or a silicone
adhesive (RTV 108 and RTV 118 from General Electric Co., Waterford,
N.Y., or Sylgard.RTM. Brand 184, Dow Corning Corp., Midland,
Mich.).
In the prototype devices, constrictions like those in both of FIGS.
2(a) ("narrow") and 2(b) ("shallow") have also been fabricated and
tested. Channels with "narrow" constrictions, as shown in FIG.
2(a), can be fabricated using a single mask and etching operation.
Channels with the "shallow" constrictions, as shown in FIG. 2(b),
require two masks and two etching operations.
All the component materials of the prototype devices except the
five minute epoxy were tested for embryo biocompatibility. In
applying the present invention, artisans will appreciate that
alternate materials may be used from those selected for the
prototype devices, but biocompatibility must always be established
through prior data and/or testing. Although many materials are
known to be compatible with or toxic to certain cells, little work
has been done to investigate the compatibility of materials used in
micro fabrication with embryos. The materials selected may also
vary depending upon the type of mammal from which the specific
embryos to be handled are taken.
In prototype testing, two-cell mouse embryos (B6SJL/F2) were
randomly assigned to and cultured on the substrata, in medium M16
(Sigma, St. Louis, Mo.) with bovine serum albumin (BSA; 4 mg/ml;
Sigma), covered with mineral oil (Sigma). All embryos were cultured
at 37.degree. C. in a 5% CO.sub.2 in air atmosphere for 96 h.
Developmental rates of embryos were examined every 24 h. The
percentage of embryos that reached the blastocyst stage for each
material was compared with the percentage from the control group.
Mouse embryos that reach the blastocyst stage, the latest possible
stage before embryo transfer, are probably not developmentally
hindered. While the absence of negative effects is not guaranteed
unless the embryos are also transferred to recipient mice and
monitored until the offspring are born, tests are commonly
concluded at the blastocyst stage for practical and economic
reasons. Most of the materials tested proved to be compatible with
the mouse embryos, including silicon wafers, SiN coatings, NOA 61,
and RTV 118. Some materials, such as the 5-minute epoxy, have not
been tested since it is only used in conceptual devices to
demonstrate mechanical and fluid principals of the invention, and
would likely not be used in production devices.
Tests were run to examine several aspects of the prototype devices.
Different tests required devices with different channel
configurations. In all the tests, a halogen bulb via optical fibers
illuminated the channel, which was viewed under a stereomicroscope.
A graduated cylinder and a stopwatch were used to determine flow
rates. Since the fluid is incompressible, the average fluid
velocity in any section of channel is just the flow rate divided by
the cross-sectional area.
Measurements of the rate of travel of the embryo for a given flow
rate occurred in a simple straight channel, 29 mm long, 162 .mu.m
deep, and 160-380 (bottom-top) .mu.m wide. The pressure gradient
was varied and the speed of the embryo was measured for each
setting. The channels were filled with phosphate buffered saline
(PBS), with and without BSA. Flasks of the medium were connected to
the channel. By adjusting the heights of the flasks, using
micrometer head translation stages, the pressure difference was
finely tuned to within 0.05 Pa. The flasks were connected to each
other by tubing between the tests to zero the pressure head. The
microfabricated prototype devices were cleaned in a hydrogen
peroxide/ammonium hydroxide/deionized water solution and new pipet
tips were adhered with epoxy before the tests were conducted. All
the tests using PBS without BSA were conducted before those with
BSA. Once the channels were filled with medium the mouse embryos
were placed in the inlet well, at the channel entrance.
Tests were run to observe the influence of channel size and shape
on the transport of embryos. For these tests, a device was
fabricated with one long, circuitous channel with 11 sections each
at one of four depths: 140, 164, 194, and 210 .mu.m. At each depth
the channel has 2 or 3 different widths. Widths, measured at the
surface of the wafer, range from 275 to 480 .mu.m. In the narrowest
segments, the embryos were geometrically constrained to travel on a
V-groove while in the other regions along a flat-bottomed channel.
The speed of travel and rotating characteristics were observed and
compared for different segments.
Observations of embryos at constrictions occurred in several
devices, with both narrow and shallow type constrictions. Embryos
were actually directed to specific constrictions. Altering the
height of the medium in each well, by adding or subtracting fluid,
tailored the pressure gradients in each branch of the channel
network. Pressure heads were adjusted by a 1 to 8 mm (10 to 80
Pa).
Just as embryos placed in medium sink to the bottom of the
container, embryos placed in microfluidic channels settle to the
bottom. In all the tests, when the medium flowed, the embryos
rolled and slid along the bottom of the channel in the direction of
flow. Often they also remained in contact with one of the side
walls of the channel. In initial tests without any surfactant in
the medium (phosphate buffered saline) the embryos appeared to roll
without slipping along the bottoms of the channels. Embryos slid or
rolled with slip along the bottoms in later tests when the medium
contained BSA (4 mg/ml).
Tests revealed that the rate of travel of an embryo in a channel
depends upon the velocity of the medium. Sometimes they stick to
the bottom of the channel when the velocity of the fluid around
them is below 50 .mu.m/s. For both media, PBS and PBS/BSA, a
pressure gradient of 0.16 Pa/mm drives the flow through the channel
at an average velocity of approximately 380 .mu.m/s. The embryos
roll at 187-250 .mu.m/s, 49 to 66% of 380 .mu.m/s. As the medium
flows more quickly, the embryos roll faster, slipping as they roll.
The actual speed of travel and the tendency to stick varies from
one embryo to the next One embryo has been observed to travel 25%
quicker than another at the same time in the same channel, in
almost the same path line. In the observed range, 150 to 1000
.mu.m/s, the velocity is linear with pressure gradient.
Results from testing the effects of channel size and shape match a
priori predictions. For a given flow rate, the average fluid
velocity and embryo speed is greater in a channel with smaller
cross-sectional area. In contrast, for a given pressure gradient,
the average fluid velocity and embryo speed is greater in a channel
with larger cross-sectional area. In both cases, embryos travel
slower on V-grooves than on flat-bottomed channels. Embryos are
also more likely to become wedged and stuck in a V-groove than on a
flat-bottomed channel.
Fluid under electroosmotic flow also caused embryos to roll through
channels. An embryo rolled along the channel bottom at
approximately 10 .mu.m/s due to the pressure driven trickle flow.
Switching on the voltage caused the mouse embryo to roll along the
channel bottom 20 .mu.m/s faster, at approximately 30 .mu.m/s,
toward the well with the negative electrode. With the voltage
polarity reversed, the embryo rolled at approximately 10 .mu.m/s in
the reverse direction. No surfactant, such as BSA was used so there
was little or no slipping. Electrical assistance, if used to move
embryos, must be applied under carefully controlled conditions to
avoid undesirable heating of the medium.
Computational fluid dynamics modeling using Fluent/UNS 4.2 (Fluent,
Inc., Lebanon, N.H.) and 2-dimensional finite element analysis of
prototype microfluidic channels with constant cross-section using
Quickfield (Tera Analysis, Inc., Tarzana, Calif.) verified the
observed flow rates and flow patterns. The embryo was modeled as a
rigid sphere. Recall that the embryo does not appear to deform
under typical conditions. To analyze the laminar flow, 1 or 2 mm
sections of channel were meshed into 10,000 to 30,000 tetragonal
elements. Once verified, computer modeling was used to determine
flow velocity profiles, design constrictions with lower pressure
drops, to observe forces on embryos retained at constrictions, and
to analyze electrically driven flows in similar channels. However,
analysis incorporating adhesion of the embryo to the channel walls
and distortion of the embryo would be significantly more complex
and was not attempted.
As discussed above, in the straight channel tests of embryo
velocity, the medium had an average velocity of 380 .mu.m/s under a
pressure gradient of 0.16 Pa/mm. Finite element analyses determined
the centerline velocity to be 815 .mu.m/s under these conditions.
When traveling in the channel, the embryo was tangent to the bottom
and one wall. Consider a 100 .mu.m diameter circle tangent to the
bottom and one side of the channel. The average velocity of the
fluid traveling through this circle when the embryo is not present
is 480 .mu.m/s. However, the embryos rolled at only 187-250
.mu.m/s, 39-52% as quickly, in both PBS and PBS/BSA media. The
velocity profile encourages the embryo to roll forward and along
the wall, which confirms visual observations. In sum, embryos roll
at 1/3 to 1/2 the speed at which fluid would flow in the same
region of the cross section.
The constrictions greatly increase fluid resistance in the
channels. Standard analytical formulas can help approximate the
resistance, but the cross-sectional shapes of the constrictions
vary with position. Three-dimensional models of the constrictions
were analyzed before masks were designed and wafers were etched.
The information gained from the finite element analyses led to
optimally-sized constrictions. The shallow constrictions, sized
individually for the geometry of the device, balance the need for
minimal flow resistance and robust fabrication. Typical
constrictions have a minimal depth of 20 .mu.m.
Studies of the placement of the embryos at the traps reveal lateral
forces on the order of 10.sup.-8 to 10.sup.-7 N force the embryo to
the side and part way up the ramp at the entrance to the shallow
constriction.
The tests revealed several interesting characteristics of
microfluidic transport, such as variations in velocity between
embryos and the tendency to roll along the bottoms of the channels,
often tangent to a side wall. However, the testing did reveal
several other issues. Electroosmotic flow may be useful to assist
embryo movement, and more so for movement of plugs of material
useful in embryo treatment and fertilization techniques. Electrical
control of fluid flow must be carefully controlled since high
voltages may harm the embryos in several ways. Even with the
embryos in sections of channel away from the electric fields, the
applied energy heatsup the medium (Joule heating) beyond
physiological temperatures and the electrolysis products alter the
pH. Note that an embryo requires about 0.029 Osmol, i.e., a
relatively high conductivity. Also, EOF is degraded in channels
with surfactant, but the embryos survive better in medium with a
surfactant, such as BSA.
Microfluidic transport free of electrical assistance offered
through gravity fed devices like that in FIG. 3(a), or through
pumped fluid pressure devices, offers an important advantage. The
medium can be easily altered with time to meet the changing
requirements of the developing embryos. Gradually changing the
composition of the medium avoids inducing stresses upon the embryo
from the abrupt environmental changes that often accompany transfer
from one petri dish to a second dish with a different medium. The
microfluidic handling of embryos by the invention is not physically
harsher than transfer with pipets and definitely less damaging than
many techniques in conventional practice including some which
pierce the outer membrane.
It is anticipated that control of fluid flow, and therefore embryo
positioning, in handling devices like that shown in FIG. 3(a) will
be handled through programmed control instruments for largely
automated devices. Alarms and warnings may be incorporated based
upon sensed conditions within an embryo handling device of the
invention. In similar fashion, monitoring of embryos with
conventional instruments applied to a handling device of the
present invention. Artisans will generally recognize that the
microfluidic embryo handling device thus forms a basic building
block upon which many useful devices may be based, and that such
devices will incorporate the essence of the present invention.
Some particular devices have been demonstrate for manipulation,
testing and handling of embryos and oocytes. A particular geometry
of microfluidic channels is shown in FIGS. 7(a) and 7(b), and has
been demonstrated to enable cumulus removal from oocytes. The
geometry is a series of gradually more constricted sections of
microfluidic channels, best seen in FIG. 7(a). Though FIG. 7(a)
shows curved constricted sections, the constricted need not be
curved. Inner surfaces of the constricted sections preferably have
protrusions in the form of teeth or serrations to aid cutting of
the cumulus as it passes. The last section in a series has
protrusions separated at a distance apart to avoid damage to the
oocyte, with the preceeding sections having protrusions spaced at
gradually smaller distances apart to cut portions of surrounding
cumulus as fluid pressure forces the oocyte through a constricted
section. In FIG. 7(a), multiple constricted sections each
individually have protrusions with equal spacing, with a downstream
section from a previous section using a lesser spacing. One could
also construct a single section in which protrusions within the
section gradually become closer together to accomplish the same
purpose of cutting deeper into the cumulus at multiple locations
without damaging the embryo. The FIGS. 7(a) and 7(b) geometry has
been used to remove cumulus from cattle oocytes. In a prototype
device, straight sections of microfluidic channel were 500 microns
wide. Five constricted sections were used. The first and largest
section included protrusions spaced at 300 microns gradually
decreasing to the fifth constricted section that was 50 microns
wide. The first four constrictions cut (a "mohawk" cut has been
observed) the cumulus as the cumulus-oocyte complex passes, forced
along by fluid flow. At the fifth constriction half of the cumulus
was suctioned off. Flow was reversed a few times to reposition the
oocyte until the other half of the cumulus was drawn through the
final constriction. Thus, appropriately dimensioned constrictions
and geometry may be used to position and condition an embryo and
surrounding structure.
A complete prototype device for cumulus removal is depicted in
FIGS. 8(a) and (b), while FIGS. 8(c)-(f) illustrate steps used in
experiments with the FIGS. 8(a) and 8(b) prototype to remove
cumulus from an oocyte. A polypropylene well is bonded to a loading
port to provide a larger fluid reservoir at the inlet shown in FIG.
8(a). An acrylic syringe connection module exit ports so that
standard syringes or other fittings can be connected to the device.
Syringes enable manual pressure control, or a syringe pump can
serve as a precise flow controller the plexi-glass and PDMS
prototypes allowed for embryo positioning throughout a channel
network and parking of the oocyte at desired locations during
testing. In addition, the prototype devices provide complete
optical access (important for embryo analysis), rapid prototyping,
and easy integration with future analysis sensors. Loading oocytes
in the device is simplified through the use of a funnel-shaped
inlet well (inset in FIG. 8(b)). The funnel shape is molded at the
entrance to the channel with the tip of the funnel connected to the
head of the channel. This wide funnel configuration allows an
oocyte complex to be easily inserted. Oocytes will typically sink
to the funnel bottom. The sloped walls guide the complex into the
channel entrance at the bottom of the funnel. This method of
loading simplifies the handling procedures because it does not
require precise lateral positioning. To manipulate the cumulus
cells into a configuration that allows for complete cumulus
removal, the complex is passed through two constricted regions
(FIG. 8(c)). These narrowed regions force the cumulus into two main
clumps at the front and back of the oocyte, as shown in FIG. 8(d).
The two constricted cumulus conditioning regions (FIG. 8(a) were
200 and 150 .mu.m wide, respectively. The cumulus gets damaged and
bunched in the conditioning regions and then flows to removal ports
(see FIGS. 8(d)-8(f)), which comprised two thin channels placed
ninety degrees from one another at a bend in the microfluidic
channel in the prototype device. The ports allow the cumulus to
enter (FIGS. 8(e) and 8(f)) while being too small for an oocyte.
Using fluid flow control, cumulus is suctioned off the oocyte,
first trough one port and then the other.
Microfluidic channels of the invention have also been used to
realize novel methods of embryo health evaluation, by analysis of
mechanical properties of the embryos. Specific mechanical
properties demonstrated to distinguish health embryos include
surface properties and deformation properties of embryos. The
tendency of an embryo to return its shape after being deformed to a
point short of permanent damage is believed to be an indicator of
health because embryos actively maintain their shape. Embryos
selectively transport ions through their membranes and the proteins
of the zona pellucida are constantly reorienting themselves to
resist external forces. Transport of ions and other
substrates/metabolites into and out of the embryonic cells is a
function of the health of the embryo. Unhealthy embryos have
degraded ability to transport ions, and a corresponding degraded
ability to return their shape after being deformed to a point that
avoids permanent damage. The control of pressure and the ability to
position embryos at constrictions offers the opportunity to deform
embryos with a control that prevents permanent deformation of
health embryos. Deformation testing has been conducted keeping
pressure gradients below 0.1 Pa/m (1 mm water/cm) and fluid
velocities at or below a few millimeters per second.
FIGS. 9(a)-9(d) illustrate a deformation evaluation of an embryo
used as an indicator of embryo viability. A constriction is sized
to cause deformation of an embryo as it passes through due to fluid
pressure. A healthy embryo better returns its shape in FIG. 9(d)
after having passed through the constriction. Alternatively, an
embryo might be deformed at constriction sized to prevent passage.
After for a period of deformation at a constriction, flow is
reversed, reversed and stopped, or stopped, allowing the embryo to
return its shape.
The precise control of pressure offered by microfluidic channels
also permits evaluation of embryo stiction to channel walls, as
healthy embryos will stick more. Thus, a healthy embryo can
quantitatively be measured to travel more slowly down a channel as
compared to an unhealthy embryo. Alternatively, distance of travel
can be similarly compared, with a healthy embryo traveling a
shorter distance under identical flow and pressure conditions.
Fluid analysis of embryos is better enabled by devices
incorporating microfluidic channels of the invention. Fluid is
collected from a downstream channel of a microfluidic channel
culturing device of the invention. In accordance with devices
formed according to the invention, the collected fluid will have
flowed past an embryo since devices of the invention avoid stagnant
conditions and sizing of the microfluidic channels causes fluid in
the channels to pass close to an embryo. When downstream fluid is
collected, it may be desirable to add a similar amount of upstream
fluid to maintain pressures and flows within a microfluidic device
from which fluid samples are being collected. An exemplary channel
configuration for fluid collection is shown in FIG. 10. An embryo
16 is positioned at a mechanical constriction. Downstream, a
collection or analysis cell 70 provides fluid which has passed in
close proximity to the positioned embryo. A side channel 72 may
provide a second fluid to aid analysis, which may be mixed in with
a main flow with the assistance of peg formations 74, though mixing
is achieved without formations as shown in previous figures.
Programmable syringe pumps can be used for removal of fluid samples
and insertion of additional upstream fluid. Alternatively, all of
fluid downstream an embryo could be collected. This fluid is then
analyzed, such as by chemical or optical analysis.
Systems of the invention can be used for complete oocyte
maturation, fertilization and embryo culturing without harsh
manipulation techniques. The ability to control fluid and position
embryos offers the chance to mature an oocyte, bring sperm or
seminal fluid into contact, and then simulate biological embryo
culturing, all within a single device of the invention. Maturation
(IVM), fertilization (IVF) and culturing (EC) require different
media, sperm capacitation and rinsing procedures. Systems of the
invention using microfluidic channels allow rapid change and
precise control of such conditions, allowing changing of the
composition of the fluidic medium over the course of hours or days
to simulate secretion and growth factor accumulation in the female
reproductive tract and embryo movement to different parts of the
tract. Medium can be controlled to flow slowly past each embryo (or
oocyte) to provide a fresh supply of nutrients. Periodic flows,
e.g. once an hour, as opposed to continuous flows, would allow a
limited build-up of beneficial autocrine and paracrine factors
without allowing waste products to accumulate. All of the current
IMF, IVF and EC techniques can be performed in a single device of
the invention such that a given oocyte-embryo does not have to be
handled or moved during a sequential IMF, IVF and EC procedure or
during any one of the individual procedures.
Systems of the invention may reduce rates of polyspermy, use
smaller numbers of spermatozoa, and incorporate a swim-up technique
to select the most motile sperm. In typical human IVF procedures,
the egg is surrounded by 50,000 to 100,000 sperm in a 0.5 to 1.0 ml
drop of medium. This number could be reduced since the microfluidic
channels provide a flow control to bring sperm and oocyte
together.
In livestock IVF, rates of polyspermy are high, about 40-70% in
pigs, for example. According to the invention, microfluidic
channels cause sperm to pass very close to oocytes, e.g., about 30
.mu.m. In addition, the time interval in which the sperm are near
the oocyte can be controlled by controlling the rate of media flow.
Alternatively, a parked oocyte positioned according to the
invention can be held very close to the point of insertion of a
comparatively small fluid packet of sperm, providing a high
concentration of sperm near the oocyte while reducing the chance of
polyspermy. Discrete plugs or boluses may deliver one to a few
sperm to create a situation analogous to the situation in vivo in
the oviduct where there are few numbers of sperm at any one time.
This ability, with a T-junction or other physical or effective
constriction to position an embryo, allows for delivery of one to a
few sperm to the proximity of the ovum to reduce the chance of
polyspermy. In addition, channel geometry being embryo scaled
allows for increased contact between the ovum and the sperm. Sperm
and "bounce" off the side walls and increase the effecfive contact
between the sperm and the egg.
Cryopreservation is another technique that would benefit from the
ability to position embryos according to the invention, and the
ability to precisely control the fluid environment around the
embryo. Cryopreservatives may be delivered to a positioned or
moving embryo in a device of the invention, which can then later be
used to reverse the cryopreservation process. Change of the fluid
can then be used to conduct maturation, fertilization, and
culturing, as described above.
While various embodiments of the present invention have been shown
and described, it should be understood that other modifications,
substitutions and alternatives are apparent to one of ordinary
skill in the art. Such modifications, substitutions and
alternatives can be made without departing from the spirit and
scope of the invention, which should be determined from the
appended claims.
Various features of the invention are set forth in the appended
claims.
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